Quantum dots have the potential to be used as contrast agents for molecular imaging in vivo, but there are many challenges in optimising such procedures, both in pre-clinical animal models and in the potential clinical applications. In particular, it is critical to obtain the optimal target-to-background contrast to achieve maximum diagnostic accuracy. However, available data are insufficient to determine the optimal parameters for this objective. Here, we explore the factors that affect imaging contrast with quantum dots, including time, dosage, emission and excitation wavelengths, and instrumentation. This includes a brief review of the published work. In addition, we present initial studies with tissue-simulating phantoms that allow the optimum excitation wavelength to be determined for visible-emitting quantum dots used to image surface or near-surface lesions, as found in endoscopic diagnosis of early cancer. We also suggest needed areas for future research for optimisation of quantum dot-based bio-imaging applications.